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Cover Page

The handle

http://hdl.handle.net/1887/85721

holds various files of this Leiden

University dissertation.

Author: Wang, L.

(2)

Chapter 6

Reagent Controlled Glycosylations for the

Assembly of Well-defined Pel

Oligosaccharides

Introduction

Pel is one of the exopolysaccharides that forms the biofilm surrounding Pseudomonas aeruginosa,

an opportunistic Gram-positive pathogen that is the major cause of morbidity and mortality in

Cystic Fibrosis patients.

[1]

Pel is a linear polysaccharide composed of 1,4-linked α-N-acetyl

galactosamine (GalNAc) and α-N-acetyl glucosamine (GlcNAc) residues, present in a ±6 : 1 ratio,

of which some of the residues have been de-acetylated to generate positively charged

galactosamine (GalN) and glucosamine (GlcN) moieties (Figure 1).

[1b]

Well-defined Pel

fragments can be used to unravel their role in biofilm formation, to study their biosynthesis and

possibly as synthetic antigens in the development of a (semi)-synthetic vaccine against P.

(3)

isolate well-defined oligosaccharides from natural sources and therefore organic synthesis is

necessary to provide these structures.

Figure 1. The repeating structures of Pel.

To assemble Pel-fragments (Figure 1), four kinds of α-bonds, α-

D

-GlcN-(1→4)-

D

-GlcN, α-

D

-GlcN-(1→4)-

D

-GalN, α-

D

-GalN-(1→4)-

D

-GlcN and α-

D

-GalN-(1→4)-

D

-GalN, have to be

constructed. Zhang et al. have recently reported an effective synthetic strategy to assembly

galactosminogalactans (GAGs), fungal polysaccharides composed of 1,4-linked α-

D

-Gal, α-

D

-GalN and α-

D

-GalNAc moieties.

[2]

For the formation of the 1,2-cis-linkage,

4,6-di-tert-butylsilylene (4,6-O-DTBS) protected GalN-donors were used to control the selectivity.

[3]

This

strategy allowed the use of galactosamine donors bearing differently masked amine

functionalities. Galactosazide and trichloroacetyl protected GalN donors were used to combine

GalN and GalNAc at pre-determined sites in the target GAG oligosaccharides. Of note, the

stereodirecting capacity of the DTBS group in GalN donors effectively overrides the neighboring

group participation by C2-particpating functionalities such as the trichloroacetamide. Thus,

DTBS-GalN donors also represent attractive building blocks for Pel-assembly. For the

stereoselective introduction of α-

D

-GlcN linkages no general solution exists, even though the

construction of this type of glycosidic linkage has attracted significant attention,

[4]

as it presents

in many important natural polysaccharides and glycoconjugates, such as heparin, heparan

sulfate,

[5]

GPI anchors and various bacterial polysaccharides.

[6]

Additive controlled

glycosylations have been introduced for the stereoselective construction of glycosidic linkages.

[7]

In these approaches the nature of the additive determines the reactivity of in situ formed

glycosylating species and the reactivity of the additive can be tuned to match the reactivity of the

glycosyl donor

[8]

and acceptor

[9]

building blocks. Chapter 2 and 3 of this Thesis have reported the

(4)

α-(1,3)-glucans from fungi cell wall.

[10]

The synthetic strategy to the oligosaccharides is built on the

use of additive controlled glycosylation reactions and the use of one single benzyl type protecting

group (Bn, PMB, Nap). For glycosylations with relatively reactive primary alcohol acceptors, the

trimethylsilyliodide (TMSI)-triphenylphosphine oxide (Ph

3

P=O) activator couple was used,

while the condensations with the less reactive secondary alcohols required the use of the

trifluoromethanesulfonic acid (TfOH)-dimethylformamide (DMF) pair. The successful

construction of multiple 1,4-α-glucosidic linkages was an incentive to explore this strategy for

the assembly of the Pel oligasaccharides. Mong and co-workers have previously described how

formamide additives can be used for the construction of 1,2-cis-GalN

3

and GlcN

3

linkages. They

introduced N-formyl-morpholine (NFM) to modulate the reactivity of tri-O-benzyl GlcN

3

and

4,6-benzylidne-GalN

3

donors and showed that the glycosylations mediated by NFM proceeded

with higher stereoselectivity than the corresponding DMF-modulated condensations.

[72n]

Because

of the stronger electron withdrawing effect of the azide group with respect to a benzyl ether,

2-azido donors are generally less reactive than their 2-O-benzyl counterparts. This lower reactivity

can be counterbalanced by the use of a somewhat more reactive additive, explaining why NFM

outperforms DMF in these glycosylations (See Scheme 1).

Scheme 1. The relative reactivity of DMF and NFM intermediates.

This Chapter describes a strategy to synthesize Pel oligosaccharides using additive-controlled

glycosylations to match the reactivities of the GlcN

3

-donor and the Pel acceptors. Because of the

relatively low nucleophilicity of the GlcN

3

-C4-OH and especially the GalN

3

-C4-OH, a new

additive is introduced that generates intermediates that are more reactive than the previously

introduced DMF and NFM-imidinium ions.

Results and discussion

First, attention was paid to the formation of α-

D

-GlcN-(1→4)-

D

-GlcN linkages. In line with

previous work, solely benzyl type protecting group (PMB, Nap, Bn) were used to generate

orthogonally protected building blocks of uniform reactivity. The synthesis of

D

-GlcN building

blocks is depicted in Scheme 2 and started from

D

-glucosamine 1. Using the diazo transfer regent

imidazole-1-sulfonyl azide hydrogen sulfate,

[11]

glucosazide was generated, which was acetylated

(5)

the fully protected GlcN

3

. Regioselective opening of the benzylidene aetal and masking of the

liberated C4-OH as a PMB-ether provided compound 4. The anomeric thio acetal in this building

block was hydrolyzed using N-iodosuccinimide in acetone/water to liberate the anomeric

hydroxyl group, which was then turned into the required N-phenyltrifluoroimidate donor 5. To

obtain the first acceptor, synthon 5 was coupled with benzyl 6-hydroxyhexanoate using the

TMSI-Ph

3

P=O conditions to provide compound 6 in 75% yield and a 5:1 α:β ratio. Deprotection of the

PMB-masked C4-alcohol using a catalytic amount of HCl in hexafluoroisopropanol (HFIP)

produced acceptor 7.

[12]

After purification by silica gel chromatography the desired α-product was

obtained as a single anomer.

(a) BF3.EtO2, TES, DCM, 3: 80%; (b) PMBCl, NaH, DMF, 4: 95%; (c) 1) NIS, acetone, H2O, 2)

2,2,2-trifluoro-N-phenylacetimidoyl chloride, Cs2CO3, acetone, 5: 83% with two steps; d) TMSI, Ph3P=O, DCM, rt, 6: 75%, α:β = 5:1; (e)

0.2 M HCl/HFIP, TES, HFIP, DCM, 7: 60%.

Scheme 2. Synthesis of donor 5 and acceptor 7.

With donor 5 and acceptor 7 in hand, DMF was investigated as an additive to control the

selectivity according to previous successful experiments. Thus, donor α-

D

-GlcN 5, acceptor 7 and

the additive were mixed in DCM with molecular sieves and cooled to -78

o

C. Next, TfOH was

added and after stirring for 0.5h, the mixture was placed at 0

o

C and allowed to stir for 24h. As

shown in Table 1, this produced the desired disaccharide product 8 with complete α-selectivity,

but the yield was only 32% (entry 1). Performing the reaction at room temperature did not lead

to erosion of stereoselectivity but only marginally improved the yield (entry 2). Likely, the low

reactivity of the azido-donor and -acceptor led to the observed poor yields and therefore NFM

was probed as an additive.

[6c]

Use of this additive provided complete α-selectivity, and raised the

yield of the condensation to 55% yield. To further improve the reaction, a more reactive additive

was sought and N-methyl-N-phenylformamide (MPF) was explored. It was expected that the

imidinium ion formed from this additive would be more reactive because the aniline-type nitrogen

would be less capable of supporting the (partial) positive charge in the ion. The reaction of donor

5 and acceptor 7 proceeded with excellent yield (91%) when performed at 0

o

C, and the

(6)

this condensation is somewhat less than the DMF or NFM mediated glyosylations, the improved

yield allows for an overall more productive reaction.

[13]

Table 1. Glycosylation between donor 5 and acceptor 7 under various additives.

entry

c(mmol/ml)

additive

T(

o

C)

product

yield

a

α:β

b

1

0.1

DMF

0

8

32%

>20:1

2

0.1

DMF

rt

8

38%

>20:1

3

0.1

NFM

rt

8

55%

>20:1

4

0.1

MPF

0

8

91%

~15:1

a Isolated yield. b The α:β ratio was determined by 1H NMR.

To probe the robustness of the MPF-mediated protocol in the synthesis of oligosaccharides, the

assembly of an all-1,2-cis linked tetrasaccharide was explored as depicted in Scheme 3. Thus,

donor 5 and acceptor 7 were coupled under the above identified reaction conditions to provide

the desired disaccharide 8. The PMB was removed using a catalytic amount of HCl to give

disaccharide acceptor 9 in 88% yield. Next, compound 9 was glycosylated with donor 5 under

the MPF-conditions to form the desired trisaccharide 10 in 83% yield and excellent

stereoselectivity (α:β > 19:1). Repetition of the deprotection and glycosylation reactions then

uneventfully provided tetrasaccharide 12. The successful assembly of this tetrasaccharide

indicates that the yield and stereoselectivity don’t decrease with the growing of the sugar chain.

(a) MPF, TfOH, DCM, -78-0oC, 8: 91%, α:β = 15:1; 10: 83%, α:β > 19:1; 12: 90%, α:β > 20:1. (b) 0.2 M HCl/HFIP, TES,

HFIP/DCM, 9: 88%; 11 78%.

(7)

Next, attention was turned to the formation of the α-GlcN-(1→4)-GalN-linkage exploring the

additives as described above. First donor 13 was coupled with acceptor 16 using DMF to provide

product 20 in low yield and poor selectivity (Table 2, entry 1). The use of NFM instead of DMF

did not improve the outcome of this glycosylation (entry 2). Likely the poor reactivity of the

sterically encumbered GalN

3

-C4-OH hampers the union of the two carbohydrate building blocks.

Table 2. Glycosylation between 2-azido Glu/Gal donors and 4-OH-2-azido Glu/Gal acceptors.

entry

donor

acceptor

c(mmol/ml)

additive

T(

o

C)

product

yield

a

α:β

b

1

13

16

0.1

DMF

0

20

23%

6:1

2

13

16

0.1

NFM

0

20

24%

6:1

3

13

16

0.1

MPF

0

20

83%

5:1

4

13

16

0.1

MPF

-10

20

43%

10:1

5

13

16

0.2

MPF

-10

20

88%

10:1

6

14

17

0.1

MPF

-10

21

88%

8:1

7

14

18

0.1

MPF

-10

22

80%

4:1

8

c

15

19

0.1

-

0

23

70%

1:0

(8)

Next, the use of MPF was explored. At 0

o

C, disaccharide 20 was obtained in high yield (83%),

but with moderate α:β-selectivity (5:1). Performing the same reaction at -10

o

C increased the

α-selectivity (α:β = 10 : 1), but led to a relatively low yield (43%, entry 4). To increase the yield of

the reaction, the concentration was raised from 0.1

M

to 0.2

M

(entry 5). This led to the formation

of the desired compound 20 with a yield of 88% and a 10:1 α:β ratio. Having defined adequate

conditions for the construction of α-GlcN-(1→4)-GlcN and α-GlcN-(1→4)-GalN linkages, the

use of MPF in combination with galactosazide donor 14 was explored for the construction of the

target α-GalN-(1→4)-GlcN and α-GalN-(1→4)-GalN linkages. Under the conditions established

above, donor 14 was coupled with glucosyl acceptor 17 to give the disaccharide 21 in excellent

yield and 8:1 α/β-stereo selectivity (Table 2, entry 6). Contrary disaccharide 22, formed from

donor 14 and galactosyl acceptor 18, was obtained in poor yield and poor selectivity (α:β = 4:1,

entry 7). Because 4,6-O-DTBS protected galactosamine donors have been shown to reliably

provide 1,2-cis-linkages, attention was next turned to this donor type. Thus, DTBS-donor 15 was

glycosylated with acceptor 19 at 0

o

C to give the desired disaccharide 23 with completely

α-selectivity (entry 8).

With conditions in hand to construct all Pel-cis linkages the synthesis of Pel hexasaccharides 24

and 25 was undertaken. A [2+2+2] strategy was designed to streamline the assembly of the

structures, building on MFP-mediated glycosylations of the GalN

3

-GlcN

3

donor 26 (Figure 2).

The procedure for the synthesis of the required building blocks 26 and 27 is depicted in Scheme

4 A and B. First donor 15 was coupled with glucoazide 19 to form disaccharide 23 as a single

anomer. Next, the silylidene ketal was cleaved with HF-pyridine, after which a benzyl ether was

regioselectively introduced under the aegis of Taylor’s borinic acid catalyst.

[14]

Protection of the

remaining C4’-OH with a naphthyl group delivered compound 30. Next the anomeric thiophenol

group was removed using N-iodosuccinimide in acetone/water, and the resulting hydroxyl group

turned into the desired N-phenyltrifluoroimidate functionality to provide donor 26. Acceptor 27

was obtained from donor 15 and acceptor 7. These two building blocks were united to

stereselectively provide disaccharide 32. Removal of the silylidene ketal and introduction of the

C6’-O-benzyl ether as described above provided 27.

(9)

With building blocks 26 and 27, the assembly of the target hexasaccharides 24 and 25 was started

(Scheme 4C). First, donor 26 was glycosylated with acceptor 27 using MPF as additive at -10

o

C

at a 0.2

M

concentration to form tetrasaccharide 34 in 89% yield as a 10:1 α/β-mixture.

(a) TfOH, DCM, 23: 70%; 32: 92%. (b) HF-pyridine, THF, 28: 98%, 33: 91%. (c) BnBr, borinic acid-catalyzed, K2CO3,

KI, CH3CN, 60 oC, 29: 96%; 27: 95%. (d) NapBr, NaH, DMF, 30: 93%. (e) NIS, acetone, H2O. (f)

2,2,2-trifluoro-N-phenylacetimidoyl chloride, Cs2CO3, acetone, 26: 83% over two steps. (g) MPF, TfOH, DCM, -10 oC, 48 h, 34: 89%, α:β

= 10:1; 36: 91%, α:β = 10:1. (h) 0.2 M HCl/HFIP, TES, HFIP/DCM, 35: 73%. (i) H2, Pd(OH)2/C, CH3COOH, THF/H2

O/t-BuOH, 24: 76%. (k) Ac2O, NaHCO3, H2O, 25: 86%.

(10)

MPF-mediated glycosylation conditions to deliver hexasaccharide 36. Reduction of the six azides and

removal of the benzyl ester and ethers were accomplished in a one-step reduction to give the

compound 24, of which the amino groups were acetylated with acetic anhydride to afford the

final compound 25.

Conclusion

In conclusion, an effective procedure to assemble Pel fragments containing 1,4-linked GalNAc

and GlcNAc residues, was developed. A [2+2+2] strategy was developed for the assembly of a

Pel-hexasaccharide in which the α-GlcN linkages were constructed in glycosylation reactions

using MPF as an additive. MPF was reported for the first time as a moderator to introduce

α-GlcN

3

linkages. The required disaccharide synthons were synthesized using a 4,6-O-DTBS

protected galactosazide donor. To probe the ability of MPF in the synthesis of oligosaccharides,

a linear glucosazide tetrasaccharide has also been assembled, through highly stereoselective

glycosylation reactions, using building blocks, solely equipped with benzyl type (Bn and PMB)

hydroxyl protecting groups. The successful synthesis reported in this Chapter show that MPF is

a valuable addition to the “additive toolbox”, which can be used to match the reactivity of donor

and acceptor building blocks to attain stereoselective glycosylation reactions.

Experimental Section

General experimental procedures

All reagents were of commercial grade and used as received. All moisture sensitive reactions were performed under an argon atmosphere. DCM used in the glycosylation reactions was dried with flamed 4Å molecular sieves before being used. Reactions were monitored by TLC analysis with detection by UV (254 nm) and where applicable by spraying with 20% sulfuric acid in EtOH or with a solution of (NH4)6Mo7O24∙4H2O (25 g/L) and (NH4)4Ce(SO4)4∙2H2O (10 g/L) in 10%

sulfuric acid (aq.) followed by charring at ~150 °C. Column chromatography was carried out using silica gel (0.040-0.063 mm). Size-exclusion chromatography was carried out using Sephadex LH-20. 1H and 13C spectra were recorded on a

Bruker AV 400 and Bruker AV 500 in CDCl3 or D2O. Chemical shifts (δ) are given in ppm relative to tetramethylsilane

as internal standard (1H NMR in CDCl

3) or the residual signal of the deuterated solvent. Coupling constants (J) are given

in Hz. All 13C spectra are proton decoupled. NMR peak assignments were made using COSY and HSQC experiments,

where applicable Clean TOCSY, HMBC and GATED experiments were used to further elucidate the structure. The anomeric product ratios were analyzed through integration of proton NMR signals.

Standard procedure

Procedure A for the glycosylation of secondary alcohols:

(11)

showed complete conversion of the acceptor. The reaction was quenched with Et3N, filtered and concentrated in vacuo.

The products were purified by size exclusion and silica gel column chromatography. Procedure B for the glycosylation of primary alcohols:

A mixture of donor (1.0 eq), acceptor (0.7 eq) (donors and acceptors co-evaporated with toluene three times), Ph3P=O (6

eq) in dry DCM were stirred over fresh flame-dried molecular sieves 3A under nitrogen. Then TMSI (1.0 eq) was added slowly in the mixture. The reaction was stirred at room temperature until TLC-analysis indicated the reaction to be complete. The solution was diluted and the reaction quenched with saturated Na2S2O3. The organic phase was washed

with water and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo. The products were purified by

size exclusion and silica gel column chromatography.

Procedure C for deprotection of the PMB and Nap protecting group:

The starting material (1 eq) was dissolved in DCM:HFIP (1:1, 0.1 M). TES (2.0 eq) and 0.2M HCl/HFIP (0.1-1eq) were added to the mixture. The reaction stirred until TLC-analysis indicated full consumption of the starting material (15min-2h). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3. The organic phase was

washed with water and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo. The product was purified

by silica gel column chromatography.

Experimental Procedures and Characterization Data of Products

For the synthesis procedure and data of known compounds 2, 3, 4see references.[15]

N-phenyl trifluoroacetimidate 2-N3-glucose donor 5: Compound 4 (9.1 g, 15.2 mmol) was

dissolved in acetone:H2O (10:1, 150 mL). N-Iodosuccinimide (NIS) (6.9 g, 30.5 mmol) was

added in one portion and the reaction was stirred at room temperature for 2 hours. The solution was diluted with DCM and the reaction was quenched with saturated aqueous Na2S2O3. Then the organic layer was washed

with water and brine. The organic layer was dried with anhydrous MgSO4, filtered and concentrated in vacuo, and the

product purified by column chromatography (pentane : ethyl acetate (EA) = 3:1). The lactol (7.2 g, 14.3 mmol) was obtained as colourless syrup. Next, the lactol was dissolved in acetone (150 mL). Cs2CO3 (7.0 g, 21.3 mmol) and

2,2,2-trifluoro-N-phenylacetimidoyl chloride (3.4 mL, 21.3 mmol) were added to the solution respectively. The reaction was stirred overnight, then quenched with Et3N, filtered and concentrated in vacuo. The product was purified by column

chromatography (pentane:EA = 40:1-20:1). Compound 5 (8.5 g, 83% over two steps, pentane:EA = 10:1, Rf = 0.45-0.55) was obtained as yellow syrup. IR (neat, cm-1) ν 697, 737, 1029, 1082, 1119, 1210, 1251, 1312, 1514, 1720, 2112 (N

3), 2872, 2912. 1H-NMR (CDCl 3, 500 MHz, 60℃) δ 7.38-7.20 (m, aromatic H), 7.11-7.06 (m, aromatic H), 6.82-6.78 (m, aromatic H), 6.37 (bs, 1 H), 5.41 (bs, 1 H), 4.92-4.80 (m), 4.74-4.69 (m), 4.60-4.48 (m), 3.96 (t, J = 10.0 Hz, 1 H), 3.90 (bd, 1 H), 3.77-3.58 (m), 3.43 (t), 3.33 (bs, 1 H). 13 C-APT (CDCl 3, 125 MHz, 60℃) δ 159.81, 159.77, 143.61, 143.49, 138.26, 138.20, 138.11, 130.31 (aromatic C), 129.66, 128.91, 128.60, 128.57, 128.54, 128.09, 128.03, 127.96, 127.94, 127.89, 127.84, 124.65, 124.57, 119.57, 114.22, 114.18 (aromatic CH), 96.23 (C-1), 94.35 (C-1), 83.29, 80.49, 77.70, 77.30, 76.36, 75.68, 75.03, 74.84, 73.92, 73.80, 73.71, 68.46, 65.81, 63.45, 55.43. Calculated for C36H35F3O6N4

(12)

Synthesis of monosaccharide 6: The reaction was carried out according to the standard procedure B. A mixture of donor 5 (1.0 g, 1.5 mmol), benzyl 6-hydroxyhexanoate (520 mg) (donors and acceptors co-evaporated with toluene three times), Ph3P=O (2.6 g, 9.3 mmol) in

dry DCM (15 mL) were stirred over fresh flame-dried molecular sieves 3A under nitrogen. Then TMSI (222 μL, 1.5 mmol) was added slowly in the mixture. The reaction was stirred at room temperature until TLC-analysis indicated the reaction to be complete. The solution was diluted and the reaction quenched with saturated Na2S2O3. The organic phase

was washed with water and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo. The products were

purified by silica gel column chromatography. Compound 6 (800 mg, 75% yield, α:β = 5:1, PE:EA = 8:1, Rf = 0.63) was obtained as a colorless syrup. IR (neat, cm-1) ν 697, 736, 1002, 1029, 1037, 1075, 1150, 1248, 1358, 1454, 1611, 1733

(C=O), 2105 (N3), 2866, 2933. 1H-NMR (CDCl3, 400 MHz) δ 7.40-7.21 (m, 15 H, aromatic H), 7.00 (bd, 2 H, aromatic

H), 6.79 (bd, 2 H, aromatic H), 5.09 (s, 2 H, PhCH2), 4.90 (d, J = 3.6 Hz, 1 H, H-1a), 4.88 (s, 2 H, PhCH2),4.71 (d, J =

10.4 Hz, 1 H, CHH), 4.63 (d, J = 12.4 Hz, 1 H, CHH), 4.49 (d, J = 12.4 Hz, 1 H, CHH), 4.43 (d, J = 10.4 Hz, 1 H, CHH), 3.975 (t, t, J = 9.6 Hz, 1 H, H-3a), 3.79-3.63 (m, 5 H, H-2a, H-4a, H-5a, H-6a, H-1ºa), 3.47-3.37 (m, 1 H, H-1ºb), 3.33 (dd,

1 H, J1 = 10.0 Hz, J2 = 2.0 Hz, H-2a), 2.36 (t, J = 7.6 Hz, 2H, H-5o), 1.70-1.58 (m, 4 H, H-2º, H-4º), 1.43-1.36 (m, 2 H,

H-3º). 13C-APT (CDCl

3, 100 MHz) δ 173.43 (C=O), 159.36, 138.09, 137.88, 130.08 (aromatic C), 129.61, 128.57, 128.50,

128.45, 128.20, 127.99, 127.96, 127.85, 127.79, 113.87 (aromatic CH), 97.91 (C-1a), 80.22 (C-3a), 78.03 (C-4a), 75.32, 74.79, 73.55 (CH2), 70.73 (C-5a), 68.30 (C-6a), 68.04 (C-1º), 66.13 (PhCH2), 63.37 (C-2a), 55.28 (OCH3), 34.161 (C-5º),

29.11 (C-2º), 25.70 (C-3º), 24.68 (C-4º). HR-MS: Calculated for C41H47N3O8 [M+NH4]+: 727.37014, found: 727.37015.

Synthesis of monosaccharide acceptor 7: The reaction was carried out according to the standard procedure C. The starting material 6 (700 mg, 0.99 m mol) was dissolved in DCM:HFIP (1:1, 0.1 M). TES (314 mL) and 0.2M HCl/HFIP (0.5 mL) were added to the mixture. The reaction stirred until TLC-analysis indicated full consumption of the starting material (15min). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3. The organic phase was washed with water and brine, dried with

anhydrous MgSO4, filtered and concentrated in vacuo. The product was purified by silica gel column chromatography.

Compound 7 (350 mg, 60% yield, PE:EA = 4:1, Rf = 0.34) was obtained as a colorless syrup. [α]D20 +59.3 (c=1, CHCl3).

IR (neat, cm-1) ν 697, 737, 1050, 1147, 1455, 1734 (C=O), 2105 (N

3), 2866, 2926, 3478. 1H-NMR (CDCl3, 400 MHz) δ

7.41-7.23 (m, 15 H, aromatic H), 5.10 (s, 2 H, PhCH2), 4.90 (d, J = 11.2 Hz, 1 H, CHH), 4.87 (d, J = 3.6 Hz, 1 H, H-1a),

4.81 (d, J = 11.2 Hz, 1 H, CHH), 4.59 (d, J = 12.0 Hz, 1 H, CHH), 4.53 (d, J = 12.0 Hz, 1 H, CHH), 3.86-3.64 (m, 6 H, H-2a, H-3a, H-4a, H-5a, H-6a, H-1ºa), 3.47-3.41 (m, 1 H, H-1ºb), 3.25 (dd, 1 H, J1 = 10.0 Hz, J2 = 2.0 Hz, H-2a), 2.37 (t,

J = 7.6 Hz, 2H, H-5o), 1.72-1.61 (m, 4 H, H-2º, H-4º), 1.47-1.37 (m, 2 H, H-3º). 13C-APT (CDCl

3, 100 MHz) δ 173.59

(C=O), 138.24, 137.86, 136.09 (aromatic C), 128.66, 128.63, 128.51, 128.27, 128.25, 128.14, 128.05, 127.85, 127.73, 127.46 (aromatic CH), 98.00 (C-1a), 79.81 (C-3a), 75.04 (C-6a), 73.72 (CH2), 72.24 (c-4a), 70.24 (c-5a), 69.76 (PhCH2),

68.07 (C-1º), 66.23 (PhCH2), 62.80 (C-2a), 34.21 (C-5º), 29.07 (C-2º), 25.74 (C-3º), 24.68 (C-4º). HR-MS: Calculated

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Synthesis of disaccharide 8: The reaction was carried out according to the standard procedure A. A mixture of donor 5 (320 mg, 0.47 mmol), acceptor 7 (185 mg, 0.31 mmol) (donors and acceptors co-evaporated with toluene three times), MPF (610 μL) in dry DCM (3 mL) were stirred over fresh flame-dried molecular sieves 3A under nitrogen. The solution was cooled to -78 ℃, after which TfOH (42 μL) was added. After 30 min, the reaction was stirred at -10 ℃ until TLC-analysis showed complete conversion of the acceptor. The reaction was quenched with Et3N, filtered and concentrated in vacuo. The product was purified by size exclusion

(DCM:MeOH = 1:1). Compound 8 (304 mg, 91% yield, α:β = 15:1, PE:EA = 4:1, Rf = 0.51) was obtained as a colorless syrup. IR (neat, cm-1) ν 697, 736, 1027, 1147, 1249, 1358, 1454, 1514, 1734 (C=O), 2103 (N

3), 2866, 2928. 1H-NMR

(CDCl3, 400 MHz) δ 7.39-7.21 (m, 25 H, aromatic H), 7.00 (bd, 2 H, aromatic H), 6.79 (bd, 2 H, aromatic H), 5.66 (d, J

= 4.0 Hz, 1 H, H-1b), 5.11 (s, 2 H, PhCH2), 4.98(d, J = 10.4 Hz, 1 H, CHH), 4.93 (d, J = 4.0 Hz, 1 H, H-1a), 4.89-4.82

(m, 3 H, 3 CHH), 4.66 (d, J = 10.0 Hz, 1 H, CHH), 4.54-4.47 (m, 3 H, 3 CHH), 4.37 (d, J = 10.4 Hz, 1 H, CHH), 4.23 (d,

J = 10.4 Hz, 1 H, CHH), 4.07 (t, J = 9.2 Hz, 1 H, H-3a), 3.98 (t, J = 9.2 Hz, 1 H, H-4a), 3.87-3.61 (m, 10 H, H-3b, H-4b,

H-5a, H-5b, H-6a, H-6ba, OCH3), 3.54-3.44 (m, 2 H, H-6bb, H-1ºa), 3.35-3.29 (m, 3 H, H-2a, H-2b, H-1ºb), 2.38 (t, J = 7.6

Hz, 2H, H-5o), 1.73-1.63 (m, 4 H, H-2º, H-4º), 1.46-1.38 (m, 2 H, H-3º). 13C-APT (CDCl

3, 100 MHz) δ 173.53 (C=O),

159.39, 138.22, 138.03, 137.84, 137.82, 136.17, 130.24 (aromatic C), 129.66, 128.65, 128.46, 128.42, 128.23, 128.13, 128.00, 127.94, 127.84, 127.78, 127.64, 127.40, 113.85 (aromatic CH), 97.78 (C-1b), 97.73 (C-1a), 80.88 (C-3a), 80.33 (C-3b), 77.82 (C-4b), 75.45, 74.73, 74.47, 73.61, 73.50 (PhCH2), 73.37 (c-4a), 71.60 (c-5b), 70.18 (C-5a), 69.09 (C-6a),

68.23 (C-6b), 67.89 (C-1º), 66.23 (PhCH2), 63.80 (C-2), 63.42 (C-2), 55.37 (OCH3), 34.24 (C-5º), 29.15 (C-2º), 25.75

(C-3º), 24.76 (C-4º). HR-MS: Calculated for C61H68N6O12 [M+NH4]+: 1094.52335, found: 1094.52388.

Synthesis of disaccharide 9: The reaction was carried out according to the standard procedure C. Compound 8 (200 mg, 0.18 mmol) was dissolved in DCM:HFIP (1:1, 0.1 M). TES (60 μL) and 0.2M HCl/HFIP (100 μL) were added to the mixture. The reaction stirred until TLC-analysis indicated full consumption of the starting material (30 min). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3. The organic phase was washed with water and brine, dried with anhydrous MgSO4, filtered and concentrated

in vacuo. The product was purified by silica gel column chromatography. Compound 9 (152 mg, 88% yield, PE:EA = 5:1,

Rf = 0.22) was obtained as a colorless syrup. [α]D20 +62.9 (c=1, CHCl3). IR (neat, cm-1) ν 697, 736, 1029, 1043, 1146,

1261, 1454, 1734 (C=O), 2105 (N3), 2868, 2926, 3491. 1H-NMR (CDCl3, 400 MHz) δ 7.42-7.20 (m, 25 H, aromatic H),

5.64 (d, J = 3.6 Hz, 1 H, H-1b), 5.11 (s, 2 H, PhCH2), 4.98 (d, J = 10.4 Hz, 1 H, CHH), 4.93 (d, J = 3.6 Hz, 1 H, H-1a),

4.89-4.82 (m, 3 H, 3 CHH), 4.55 (d, J = 12.0 Hz, 1 H, CHH), 4.51 (d, J = 12.0 Hz, 1 H, CHH), 4.08 (dd, J1 = 8.8 Hz, J2

= 10.0 Hz, 1 H, H-3a), 3.99 (t, J = 8.8 Hz, 1 H, H-4a), 3.86-3.65 (m, 7 H, H-3b, H-4b, H-5a, H-5b, H-6b, H-6aa),

3.53-3.44 (m, 2 H, H-6ab, H-1ºa), 3.40-3.33 (m, 2 H, H-2a, H-1ºb), 3.24 (dd, J1 = 3.6 Hz, J2 = 10.0 Hz, 1 H, H-2b), 2.68 (bs, 1

H, OH), 2.38 (t, J = 7.6 Hz, 2H, H-5o), 1.73-1.64 (m, 4 H, H-2º, H-4º), 1.47-1.39 (m, 2 H, H-3º). 13C-APT (CDCl 3, 100

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69.01 (C-6b), 68.23 (C-1º), 66.26 (PhCH2), 63.78 (C-2a), 62.75 (C-2b), 34.26 (C-5º), 29.15 (C-2º), 25.76 (C-3º), 24.77

(C-4º). HR-MS: Calculated for C53H60N6O11 [M+NH4]+: 974.46583, found: 974.46576.

Synthesis of trisaccharide 10: The reaction was carried out according to the standard procedure A. A mixture of donor 5 (160 mg, 0.24 mmol), acceptor 9 (150 mg, 0.16 mmol) (donors and acceptors co-evaporated with toluene three times), MPF (307 mL) in dry DCM (1.5 mL) were stirred over fresh flame-dried molecular sieves 3A under nitrogen. The solution was cooled to -78 ℃, after which TfOH (300 μL) was added. After 30 min, the reaction was stirred at -10 ℃ until TLC-analysis showed complete conversion of the acceptor. The reaction was quenched with Et3N, filtered and concentrated in vacuo. The product was purified by size exclusion (DCM:MeOH = 1:1). Compound 10

(186 mg, 83% yield, α:β > 19:1, PE:EA = 4:1, Rf = 0.40) was obtained as a colorless syrup. [α]D20 +75.8 (c=1, CHCl3).

IR (neat, cm-1) ν 697, 736, 1029, 1147, 1249, 1359, 1454, 1514, 1734 (C=O), 2106 (N

3), 2866, 2932. 1H-NMR (CDCl3,

400 MHz) δ 7.39-7.21 (m, 35 H, aromatic H), 7.00 (bd, 2 H, aromatic H), 6.79 (bd, 2 H, aromatic H), 5.69 (d, J = 3.6 Hz, 1 H, H-1), 5.67 (d, J = 3.6 Hz, 1 H, H-1), 5.11 (s, 2 H, PhCH2), 5.02-4.82 (m, 7 H, 6 CHH, H-1a), 4.66 (d, J = 10.0 Hz, 1 H, CHH), 4.56-4.46 (m, 3 H, 3 CHH), 4.39-4.33 (m, 2 H, 2 CHH), 4.26 (d, J = 12.0 Hz, 1 H, CHH), 4.18 (d, J = 12.0 Hz, 1 H, CHH), 4.14-3.98 (m, 4 H), 3.90-3.59 (m, 11 H), 3.56-3.44 (m, 3 H), 3.37-3.24 (m, 3 H), 2.38 (t, J = 7.6 Hz, 2H, H-5o), 1.73-1.63 (m, 4 H, H-2º, H-4º), 1.47-1.39 (m, 2 H, H-3º). 13C-APT (CDCl 3, 100 MHz) δ 173.49 (C=O), 159.36, 138.27, 138.17, 137.99, 137.79, 137.75, 137.55, 136.14, 130.25 (aromatic C), 129.61, 128.62, 128.57, 128.55, 128.42, 128.31, 128.26, 128.13, 127.92, 127.86, 127.81, 127.70, 127.63, 127.52, 127.41, 127.33, 113.82 (aromatic CH), 97.85, 97.73, 97.42 (C-1a, 1b and 1c), 81.04, 80.74, 79.89 (C-3a, 3b and 3c), 77.73 (C-4c), 75.26, 74.68, 74.57, 74.24, 73.57, 73.50 (PhCH2), 72.96, 72.46 4a and 4b), 71.45, 71.12, 70.16 (c-5a, 5b and 5c), 68.90, 68.65 (2 C-6), 68.24 1º), 67.73

(C-6), 66.20 (PhCH2), 63.89, 63.63, 63.13 (C-2a, 2b and 2c), 55.34 (OCH3), 34.21 (C-5º), 29.13 (C-2º), 25.73 (C-3º), 24.74

(C-4º). HR-MS: Calculated for C81H89N9O16 [M+NH4]+: 1461.67655, found: 1461.67594.

Synthesis of trisaccharide acceptor 11: The reaction was carried out according to the standard procedure C. The starting material 10 (320 mg, 0.22 mmol) was dissolved in DCM:HFIP (1:1, 0.1 M). TES (71 μL) and 0.2M HCl/HFIP (110 μL) were added to the mixture. The reaction stirred until TLC-analysis indicated full consumption of the starting material (15min). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3.

The organic phase was washed with water and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo.

The product was purified by silica gel column chromatography. Compound 11 (230 mg, 78% yield) was obtained as a colorless syrup. [α]D20 +51.0 (c=3mg/mL, CHCl3). IR (neat, cm-1) ν 697, 737, 1028, 1148, 1454, 1736 (C=O), 2106 (N3),

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137.81, 137.61, 137.53, 136.17 (aromatic C), 138.69, 128.67, 128.61, 128.55, 128.49, 128.36, 128.31, 128.13, 128.09, 128.00, 127.95, 127.92, 127.79, 127.73, 127.54, 127.48, 127.26 (aromatic CH), 97.78, 97.72, 97.42 (C-1a, 1b and 1c), 81.13, 80.77, 79.14 (C-3a, 3b and 3c), 75.02, 74.59, 74.27, 73.71, 73.53, 73.38 (PhCH2), 73.04, 72.95, 72.25 (C-4a, 4b

and 4c), 71.09, 70.31, 70.18 (c-5a, 5b and 5c), 70.02, 68.92, 68.55 (C-6a, 6b and 6c), 68.30 (C-1º), 66.26 (PhCH2), 63.93,

63.66, 62.46 (C-2a, 2b and 2c), 34.27 (C-5º), 29.17 (C-2º), 25.77 (C-3º), 24.79 (C-4º). HR-MS: Calculated for C73H81N9O15 [M+NH4]+: 1341.61904, found: 1341.61923.

Synthesis of tetrasaccharide 12: The reaction was carried out according to the standard procedure A. A mixture of donor 5 (40 mg, 0.06 mmol), acceptor 11 (35 mg, 0.03 mmol) (donors and acceptors co-evaporated with toluene three times), MPF (52 μL) in dry DCM (0.3 mL) were stirred over fresh flame-dried molecular sieves 3A under nitrogen. The solution was cooled to -78 ℃, after which TfOH (5 μL) was added. After 30 min, the reaction was stirred at -10 ℃ until TLC-analysis showed complete conversion of the acceptor. The reaction was quenched with Et3N, filtered and concentrated in vacuo. The product was purified by size exclusion (DCM:MeOH = 1:1).

Compound 12 (43 mg, 90% yield, α:β > 20:1) was obtained as a colorless syrup. [α]D20 +94.8 (c=1, CHCl3). IR (neat, cm -1) ν 697, 737, 1029, 1148, 1251, 1359, 1454, 1514, 1735 (C=O), 2106 (N

3), 2868, 2928. 1H-NMR (CDCl3, 400 MHz) δ

7.42-7.15 (m, 45 H, aromatic H), 7.00 (bd, 2 H, aromatic H), 6.79 (bd, 2 H, aromatic H), 5.70-5.67 (m, 3 H, H-1b, 1c, 1d), 5.11 (s, 2 H, PhCH2), 5.02-4.87 (m, 8 H, 7 CHH, H-1a), 4.81 (d, J = 10.4 Hz, 1 H, CHH), 4.66 (d, J = 10.4 Hz, 1 H, CHH), 4.54 (s, 2 H, PhCH2), 4.65 (d, J = 12.0 Hz, 1 H, CHH), 4.38-4.28 (m, 4 H, 4 CHH), 4.22-4.00 (m, 8 H), 3.90-3.59 (m, 15 H), 3.52-3.44 (m, 3 H), 3.39-3.34 (m, 7 H), 2.38 (t, J = 7.6 Hz, 2H, H-5o), 1.73-1.64 (m, 4 H, H-2º, H-4º), 1.47-1.38 (m, 2 H, H-3º). 13C-APT (CDCl 3, 100 MHz) δ 173.54 (C=O), 159.41, 138.31, 138.23, 138.00, 137.84, 137.78, 137.55, 136.17, 130.29 (aromatic C), 129.66, 128.66, 128.65, 128.61, 128.59, 128.48, 128.45, 128.40, 128.33, 128.30, 128.16, 128.02, 127.98, 127.93, 127.87, 127.84, 127.74, 127.71, 127.58, 127.52, 127.43, 127.34, 113.86 (aromatic CH), 97.91, 97.76, 97.52, 97.48 (C-1a, 1b, 1c and 1d), 80.96, 80.88, 80.79, 79.95 (C-3a, 3b, 3c and 3d), 77.76 (C-4), 75.30, 74.73, 74.37, 74.31, 73.60, 73.56, 73.54, 73.51 (PhCH2), 73.08 (C-4), 72.38 (C-4), 72.09 (C-4), 71.48, 71.20, 71.0570.20 (c-5a,

5b, 5c and 5d), 68.86, 68.61, 68.29 (3 C-6), 68.29 (C-1º), 67.75 (C-6), 66.25 (PhCH2), 63.81, 63.70, 63.60, 63.19 (C-2a,

2b, 2c and 2d), 55.39 (OCH3), 34.26 (C-5º), 29.17 (C-2º), 25.77 (C-3º), 24.79 (C-4º).

Synthesis of N-phenyl trifluoroacetimidate 2-N3

-glucose donor 13: Compound S1 (8.5 g, 15 mmol) was dissolved in acetone:H2O (10:1, 150 mL).

N-Iodosuccinimide (NIS) (6.7 g, 30 mmol) was added in one portion and the reaction was stirred at room temperature for 2 hours. The solution was diluted with DCM and the reaction was quenched with saturated aqueous Na2S2O3. Then the

organic layer was washed with water and brine. The organic layer was dried with anhydrous MgSO4, filtered and

concentrated in vacuo, and the product purified by column chromatography (pentane : ethyl acetate (EA) = 3:1). The lactol (6.1 g, 13 mmol) was obtained as colorless syrup. Next, the lactol was dissolved in acetone (150 mL). Cs2CO3 (6.4

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was purified by column chromatography (pentane:EA = 40:1-20:1). Compound 13 (7.3 g, 87%) was obtained as yellow syrup. IR (neat, cm-1) ν 694, 734, 1027, 1073, 1116, 1150, 1208, 1312, 1361, 1452, 1490, 1497, 1598, 1717, 2110 (N 3), 2869, 3032. 1H-NMR (CDCl 3, 500 MHz, 60℃) δ 7.52-6.81 (m, aromatic H), 6.37 (bs, 1 H, H-1α), 5.43 (bs, 1 H, H-1β), 4.89-4.76 (m, CHH), 4.60-4.48 (m, CHH), 3.98 (t, J = 9.5 Hz, 1 H), 3.91 (bd, 1 H), 3.80-3.59 (m), 3.46 (t), 3.36 (bs, 1 H). 13 C-APT (CDCl 3, 125 MHz, 60℃) δ 143.61, 143.50, 138.23, 138.16, 138.14, 138.12, 138.06 (aromatic C), 129.54, 128.92, 128.79, 128.62, 128.58, 128.54, 128.17, 128.14, 128.06, 128.04, 128.00, 127.97, 127.95, 127.91, 127.86, 126.54, 124.68, 124.60, 120.78, 119.59 (aromatic CH), 96.24 (C-1), 94.37 (C-1), 83.28, 80.49, 77.99, 77.62, 76.33, 75.74, 75.73, 75.37, 75.18, 73.90, 73.82, 73.74, 68.45, 65.83, 63.48. HR-MS: Calculated for C35H33F3O5N4 [M-[O(C=NPh)CF3]+OH+Na]+:

498.19994, found: 498.19848.

Synthesis of N-phenyl trifluoroacetimidate 2-N3-galactose

donor 14: Compound S2 (3.7 g, 6.0 mmol) was dissolved in acetone:H2O (10:1, 150 mL). N-Iodosuccinimide (NIS) (2.7 g,

12 mmol) was added in one portion and the reaction was stirred at room temperature for 2 hours. The solution was diluted with DCM and the reaction was quenched with saturated aqueous Na2S2O3. Then the organic layer was washed with water and brine. The organic layer was dried with anhydrous

MgSO4, filtered and concentrated in vacuo, and the product purified by column chromatography (pentane:EA = 3:1). The

lactol was obtained as colourless syrup. Next, the lactol was dissolved in acetone. Cs2CO3 (3.0 g, 9 mmol) and

2,2,2-trifluoro-N-phenylacetimidoyl chloride (1.5 mL, 9 mmol) were added to the solution respectively. The reaction was stirred overnight, then quenched with Et3N, filtered and concentrated in vacuo. The product was purified by column

chromatography (pentane:EA = 40:1-20:1). Compound 14 (3.3 g, 86%) was obtained as yellow syrup. IR (neat, cm-1) ν

695, 734, 751, 986, 1027, 1153, 1316, 1364, 1454, 1490, 1497, 1590, 1717, 2114 (N3), 2870, 2915. 1H-NMR (CDCl3, 500 MHz, 60℃) δ 7.56-6.79 (m, aromatic H), 6.35 (bs, 1 H, H-1), 5.49 (bs, 1 H, H-1), 5.28 (d), 4.90-4.84 (m, CHH), 4.78-4.31 (m), 4.15-3.83 (m), 3.76 (dd), 3.65-3.31 (m). 13 C-APT (CDCl 3, 125 MHz, 60℃) δ 143.48, 143.36, 138.46, 138.30, 138.25, 138.17, 138.14, 137.73, 137.69, 137.64, 137.57, 137.38, 137.33, 135.19 (aromatic C), 129.48, 128.83, 128.67, 128.63, 128.60, 128.56, 128.48, 128.46, 128.43, 128.37, 128.34, 128.19, 128.17, 128.15, 128.14, 128.07, 128.03, 128.00, 127.95, 127.91, 126.48, 124.46, 120.62, 119.38 (aromatic CH), 96.53 (C-1), 92.46 (C-1), 80.89, 80.69, 77.44, 75.05, 74.93, 74.77, 74.75, 74.65, 73.81, 73.67, 73.65, 73.62, 73.53, 72.89, 72.67, 72.64, 72.46, 72.43, 72.30, 72.24, 71.90, 69.73, 69.30, 68.74, 68.27, 68.05, 64.67, 62.24, 60.43, 59.21. HR-MS: Calculated for C35H33F3N4O5 [M+Na]+: 669.22953,

found: 669.22913.

Synthesis of N-phenyl trifluoroacetimidate 2-N3

-galactose donor 15: NIS (9.15 g, 40.68 mmol) was added to the solution of compound S3 (18 g, 31.3 mmol) in Acetone/H2O (210 ml/72ml) at 0 oC. The reaction was

slowly warmed to room temperature and stirred until TLC-analysis indicated full consumption of the starting material (± 1h). Then the mixture was diluted with DCM and washed with saturated Na2S2O3 and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo. The lactol

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24.33 mmol) in 140 ml acetone. The mixture was stirred at 0 oC for 15 minutes. Then CF

3C(=NPh)Cl (6.06 g, 29.2 mmol)

was added to the solution. which was slowly warmed to room temperature and stirred overnight. The reaction was quenched with Et3N and concentrated in vacuo. The product 15 was purified by silica gel column chromatography

(PE:Et2O = 30:1 – 10:1). Compound 15 (13.3 g, a/b = 2:1, 90% yield, PE: Et2O = 10:1, Rf = 0.45-0.55) was obtained as

white solid. a isomer: 1H-NMR (CDCl

3, 400 MHz) δ 7.50 – 7.24 (m, 7H, aromatic H), 7.15 – 7.05 (m, 1H, aromatic H),

6.84 (d, J = 7.7 Hz, 2H, aromatic H), 6.47 (bs, 1H, H-1), 4.78 (d, J = 11.4 Hz, 1H, CH2Ph), 4.69 (d, J = 11.4 Hz, 1H,

CH2Ph), 4.63 (s, 1H, H-4), 4.22 (q, J = 12.8 Hz, 2H. H-6), 4.10 (t, J = 6.3 Hz, 1H, H-2), 3.89 (d, J = 9.5 Hz, 1H, H-3),

3.76 (s, 1H, H-5), 1.09-1.02 (m, 18H, CH3). 13C NMR (100 MHz, CDCl3) δ 143.29, 137.45, 128.74, 128.56, 128.01,

127.91, 124.40, 119.35 (aromatic C/CH), 94.73 (C-1), 76.04 (C-3), 70.71 (CH2Ph), 69.89 5), 69.16 4), 66.76

(C-6), 57.71 (C-2), 27.59 (CH3), 27.23 (CH3), 23.38 (C-Si), 20.73 (C-Si). b isomer: 1H-NMR (CDCl3, 400 MHz) δ 7.48 –

7.25 (m, 7H, aromatic H), 7.14 – 7.04 (m, 1H, aromatic H), 6.85 (d, J = 7.7 Hz, 2H, aromatic H), 5.50 (bs, 1H, H-1), 4.77 (d, J = 11.9 Hz, 1H, CH2Ph), 4.66 (d, J = 11.9 Hz, 1H, CH2Ph), 4.43 (s, 1H, H-5), 4.19 (s, 2H, H-6), 4.02 (s, 1H, H-4),

3.30 (s, 2H, H-2, 3), 1.15 – 1.00 (m, 18H, CH3). 13C NMR (100 MHz, CDCl3) δ 143.45, 137.54, 128.83, 128.71, 128.17,

127.97, 124.48, 119.42 (aromatic C/CH), 95.82 (C-1), 79.55 (C-3), 72.18 (C-2), 70.99 (CH2Ph), 68.57 5), 66.84

(C-6), 60.79 (C-4), 27.72 (CH3), 27.42 (CH3), 23.55 (C-Si), 20.89 (C-Si). HR-MS: Calculated for C29H37F3N4O5Si [M+Na]+:

629.2383, found: 629.2376. Synthesis of acceptor 16

Donor 15 (620 mg, 1.0 mmol) and 2-azidoethanol (178 mg, 2.0 mmol) were dissolved in DCM, cooled to 0 oC and TfOH

(15 μL, 0.1 mmol) was added. The reaction was stirred at 0 ℃ until TLC-analysis showed complete conversion of the donor. The reaction was quenched with Et3N after completed checking by TLC, filtered and concentrated in vacuo.

Compound S11 (370 mg, 73%) was obtained with full α-selectivity. Then compound S11 was dissolved in THF. HF-pyridine was added to the solution. After TLC-analysis showed complete consumption of the starting material, the reaction was quenched with saturated NaHCO3. The mixture was diluted with ethyl acetate, washed with H2O and brine, dried

with anhydrous MgSO4, filtered, concentrated in vacuo. Crude compound S12, K2CO3, KI, and borinic acid-catalyzed

were mixed in CH3CN, and then BnBr was added in the solution. The reaction was stirred at 60 ℃ until TLC-analysis

showed complete conversion of the starting material. The reaction was quenched with H2O after completed checking by

TLC, filtered and concentrated in vacuo, purified by column chromatography. Compound 16 (280 mg, 84% yield over two steps) was obtained as colorless syrup. [α]D20 +89.9 (c=1, CHCl3). IR (neat, cm-1) ν 698, 738, 1052, 1096, 1146, 1454,

2108 (N3), 2873, 2923, 3483. 1H-NMR (CDCl3, 500 MHz) δ 7.40-7.28 (m, 10 H, aromatic H), 4.95 (d, J = 3.5 Hz, 1 H,

H-1a), 4.71 (d, J = 11.5 Hz, 1 H, CHH), 4.68 (d, J = 11.5 Hz, 1 H, CHH), 4.60 (d, J = 12.0 Hz, 1 H, CHH), 4.57 (d, J = 12.0 Hz, 1 H, CHH), 4.12 (t, J = 1.5 Hz, 1 H, H-4a), 3.98 (t, J = 6.0 Hz, 1 H, H-5a), 3.93 (dd, 1 H, J1 = 10.5 Hz, J2 = 3.0

Hz, H-3a), 3.90-3.86 (m, 1 H, H-1ºa), 3.77-3.63 (m, 4 H, H-2a, H-6ª, H-1ºb), 3.57-3.52 (m, 1 H, H-2ºa), 3.37-3.33 (m, 1

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128.78, 128.58, 128.35, 128.23, 127.92, 127.83 (aromatic CH), 98.48 (C-1a), 76.02 (C-3a), 73.78, 72.13 (CH2), 69.60

(C-6a), 69.21 (C-5a), 67.24 (C-1º), 66.83 (C-4a), 58.98 (C-2a), 50.76 (C-2º). HR-MS: Calculated for C22H26O5N6 [M+NH4]+:

472.23029, found: 472.23003. Synthesis of acceptor 17

Donor 5 (820 mg, 1.2 mmol), isopropanol (200 μL, 2.6 mmol) and Ph3P=O (2 g) were dissolved in DCM (12 mL), and

TMSI (173 μL) was added at room temperature. The reaction was stirred at rt until TLC-analysis showed complete conversion of the donor. The reaction was quenched with Et3N after completed checking by TLC, filtered and

concentrated in vacuo, purified by column chromatography. Compound S13 was obtained with α:β = 5:1. Then compound S13 was dissolved in DCM/HFIP (1.5 mL: 1.5 mL). TES (380 μL) and 0.2M HCl/HFIP (600 μL) were added to the mixture. The reaction stirred until TLC-analysis indicated full consumption of the starting material (30min). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3. The organic phase was washed with

water and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo. The product was purified by silica gel

column chromatography. Compound 17 (240 mg, 47% yield over two steps) was obtained as colorless syrup. [α]D20 +83.4

(c=1, CHCl3). IR (neat, cm-1) ν 697, 735, 1029, 1047, 1120, 1454, 2105 (N3), 2920, 2974, 3476. 1H-NMR (CDCl3, 400

MHz) δ 7.40-7.20 (m, 10 H, aromatic H), 4.97 (d, J = 3.6 Hz, 1 H, H-1a), 4.88 (d, J = 11.2 Hz, 1 H, CHH), 4.78 (d, J = 11.2 Hz, 1 H, CHH), 4.57 (d, J = 12.0 Hz, 1 H, CHH), 4.50 (d, J = 12.0 Hz, 1 H, CHH), 3.93-3.82 (m, 3 H, H-3a, H-5a, H-1º), 3.73-3.61 (m, 3 H, H-4a, H-6a), 3.18 (dd, 1 H, J1 = 10.0 Hz, J2 = 3.6 Hz, H-2a), 2.76 (bs, 1 H, OH), 1.23, (d, J =

8.4 Hz, 3 H, CH3), 1.21 (d, J = 8.4 Hz, 3 H, CH3). 13C-APT (CDCl3, 100 MHz) δ 138.20, 137.80 (aromatic C), 128.54,

128.39, 128.00, 127.90, 127.72, 127.59 (aromatic CH), 96.37 (C-1a), 79.64 (C-3a), 74.85, 73.56 (CH2), 72.06 (C-4a),

70.83 (C-1º), 70.12 (C-5a), 69.65 (C-6a), 62.51 (C-2a), 23.23 (CH3), 21.48 (CH3). HR-MS: Calculated for C23H29O5N3

[M+NH4]+: 445.24455, found: 445.24441.

Synthesis of acceptor 18

Donor 15 (2.77 g, 4.6 mmol) and isopropanol were dissolved in DCM (40 mL), cooled to 0 oC and TfOH (40 μL) was

added. The reaction was stirred at 0 ℃ until TLC-analysis showed complete conversion of the donor. The reaction was quenched with Et3N after completed checking by TLC, filtered and concentrated in vacuo. Compound S14 was obtained

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NaHCO3. The mixture was diluted with ethyl acetate, washed with H2O and brine, dried with anhydrous MgSO4, filtered,

concentrated in vacuo, purified by column chromatography. Compound S14 (1.45 g) was obtained with 94% yield over two steps. Then compound S14 (665 mg, 1.97 mmol), K2CO3 (293 mg), KI (327 mg), and borinic acid-catalyzed (44 mg)

were mixed in CH3CN (20 mL), and then BnBr was added in the solution. The reaction was stirred at 60 ℃ until

TLC-analysis showed complete conversion of the starting material. The reaction was quenched with H2O after completed

checking by TLC, filtered and concentrated in vacuo, purified by column chromatography. Compound 17 (745 mg, 80% yield) was obtained as colorless syrup. [α]D20 +102.7 (c=1, CHCl3). IR (neat, cm-1) ν 698, 737, 1052, 1454, 2108 (N3),

2892, 2926. 2972. 1H-NMR (CDCl

3, 400 MHz) δ 7.42-7.27 (m, 10 H, aromatic H), 5.02 (d, J = 3.6 Hz, 1 H, H-1a), 4.71

(bs, 2 H, PhCH2), 4.58 (bs, 2 H, PhCH2), 4.15 (t, J = 1.6 Hz, 1 H, H-4a), 4.01 (bt, 1 H, H-5a), 3.95-3.89 (m, 2 H, H-3a,

H-1º), 3.76 (dd, 1 H, J1 = 10.0 Hz, J2 = 6.0 Hz, H-6aa), 3.70-3.62 (m, 2 H, H-6ab, H-2a), 2.60 (bs, 1 H, OH), 1.23 (d, 3 H,

J = 10.4 Hz, CH3), 1.21 (d, 3 H, J = 10.4 Hz, CH3). 13C-APT (CDCl3, 100 MHz) δ 138.03, 137.45 (aromatic C), 128.76,

128.55, 128.29, 128.12, 127.87, 127.77 (aromatic CH), 96.68 (C-1a), 76.14 (C-3a), 73.77, 71.96 (CH2), 70.90 (C-1º),

69.58 (C-6a), 68.73 (C-5a), 66.79 (C-4a), 59.02 (C-2a), 23.34 (CH3), 21.63 (CH3). HR-MS: Calculated for C23H29O5N3

[M+NH4]+: 445.24455, found: 445.24455.

Synthesis of disaccharide 20: The reaction was carried out according to the standard procedure A. A mixture of donor 13 (146 mg, 0.22 mmol), acceptor 16 (50 mg, 0.11 mmol) (donors and acceptors co-evaporated with toluene three times), MPF (216 μL) in dry DCM were stirred over fresh flame-dried molecular sieves 3A under nitrogen. The solution was cooled to -78 ℃, after which TfOH (19 μL) was added. After 30 min, the reaction was stirred at -10 ℃ until TLC-analysis showed complete conversion of the acceptor. The reaction was quenched with Et3N, filtered and concentrated in vacuo. The product was purified by size exclusion

(DCM:MeOH = 1:1). Compound 20 (86 mg, 88%, α:β = 10:1) was obtained as a colorless syrup. IR (neat, cm-1) ν 697,

736, 1027, 1046, 1093, 1127, 1150, 1259, 1359, 1454, 2105 (N3), 2869, 2923. 1H-NMR (CDCl3, 400 MHz) δ 7.40-7.05

(m, 25 H, aromatic H), 4.99 (bt, 2 H, H-1a and H-1b), 4.90-4.76 (m, 3 H, 3 CHH), 4.69 (d, J = 10.8 Hz, 1 H, CHH), 4.63 (d, J = 10.8 Hz, 1 H, CHH), 4.59-4.53 (m, 2 H, 2 CHH), 4.39 (bt, 2 H, 2 CHH), 4.31 (d, J = 2.4 Hz, 1 H), 4.13-3.49 (m, 13 H), 3.39-3.29 (m, 2 H), 3.22 (dd, J1 = 12.4 Hz, J2 = 2.0 Hz, 1 H), 2.96 (dd, J1 = 10.8 Hz, J2 = 2.0 Hz, 1 H), 4.48 (d, J1

= 10.8 Hz, J2 = 1.6 Hz, 1 H). 13C-APT (CDCl3, 100 MHz) δ 138.05, 137.75, 137.654, 137.49 (aromatic C), 128.62, 128.51,

128.42, 128.35, 128.18, 128.07, 128.06, 127.88, 127.82, 127.78, 127.75, 127.70, 127.22 (aromatic CH), 98.92 (C-1), 98.53 (C-1), 80.15, 78.12, 75.58, 75.42, 74.91, 73.66, 73.34, 73.28, 72.00, 70.94, 69.60, 67.33, 67.26, 67.02, 64.03, 59.36, 50.71. HR-MS: Calculated for C49H53O9N9 [M+NH4]+: 929.43045, found: 929.43039.

Synthesis of disaccharide 21: The reaction was carried out according to the standard procedure A. A mixture of donor 14 (77 mg, 0.12 mmol), acceptor 17 (34 mg, 0.08 mmol) (donors and acceptors co-evaporated with toluene three times), MPF (156 μL) in dry DCM were stirred over fresh flame-dried molecular sieves 3A under nitrogen. The solution was cooled to -78 ℃, after which TfOH (8 μL) was added. After 30 min, the reaction was stirred at -10 ℃ until TLC-analysis showed complete conversion of the acceptor. The reaction was quenched with Et3N,

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mg, 88% yield, α:β = 8:1) was obtained as a colorless syrup. [α]D20 +85.8 (c=1, CHCl3). IR (neat, cm-1) ν 697, 736, 986, 1037, 1117, 1209, 1261, 1454, 2106 (N3), 2870, 2925. 1H-NMR (CDCl3, 400 MHz) δ 7.41-7.19 (m, 25 H, aromatic H), 5.64 (d, J = 3.6 Hz, 1 H, H-1a), 5.03 (d, J = 3.6 Hz, 1 H, H-1b), 4.96 (d, J = 10.0 Hz, 1 H, CHH), 4.91 (d, J = 10.0 Hz, 1 H, CHH), 4.81 (d, J = 11.2 Hz, 1 H, CHH), 4.67 (d, J = 11.2 Hz, 1 H, CHH), 4.61 (d, J = 11.2 Hz, 1 H, CHH), 4.56 (d, J = 12.4 Hz, 1 H, CHH), 4.48 (d, J = 11.2 Hz, 1 H, CHH), 4.44 (d, J = 12.4 Hz, 1 H, CHH), 4.29 (d, J = 11.6 Hz, 1 H, CHH), 4.22 (d, J = 11.6 Hz, 1 H, CHH), 4.07 (dd, J = 8.0, 10.0 Hz, 1 H, H-3b), 3.98-3.78 (m, 7 H), 3.72-3.63 (m, 2 H, H-6), 3.48-3.37 (m, 2 H, H-6), 3.29 (dd, J = 3.6, 10.0 Hz, 1H, H-2b), 1.28 (d, J = 6.4 Hz, 1 H, CH3), 1.24 (d, J = 6.4 Hz, 1 H, CH3). 13C-APT (CDCl3, 100 MHz) δ 138.44, 138.24, 137.89, 137.60 (aromatic C), 128.62, 128.57, 128.50, 128.42, 128.37, 128.33, 128.03, 127.92, 127.89, 127.85, 127.81, 127.54, 127.45 (aromatic CH), 98.04 (C-1a), 96.16 (C-1b), 80.78 (C-3b), 77.58 (C-3a), 74.89, 74.48 (PhCH2), 73.94 (C-2a), 73.57, 73.18 (PhCH2), 72.94 (C-4b), 72.16 (PhCH2), 71.08 (C-4a), 70.18 (C-5b), 70.07 (C-5a), 69.51 (C-6), 68.52 (C-6), 63.63 (C-2b), 59.75 (C-1º), 23.38 (CH3), 21.65 (CH3). HR-MS:

Calculated for C50H56O9N6 [M+NH4]+: 902.44470, found: 902.44482.

Synthesis of disaccharide 22: The reaction was carried out according to the standard procedure A. A mixture of donor 14 (77 mg, 0.12 mmol), acceptor 18 (34 mg, 0.08 mmol) (donors and acceptors co-evaporated with toluene three times), MPF (156 μL) in dry DCM were stirred over fresh flame-dried molecular sieves 3A under nitrogen. The solution was cooled to -78 ℃, after which TfOH (8 μL) was added. After 30 min, the reaction was stirred at -10 ℃ until TLC-analysis showed complete conversion of the acceptor. The reaction was quenched with Et3N, filtered

and concentrated in vacuo. The product was purified by size exclusion (DCM:MeOH = 1:1). Compound 22 (56 mg, 80% yield, α:β = 4:1) was obtained as a colorless syrup. IR (neat, cm-1) ν 697, 737, 1050, 1097, 1122, 1258, 1454, 2108 (N

3),

2869, 2928. 1H-NMR (CDCl

3, 400 MHz) δ 7.43-7.12 (m, 25 H, aromatic H), 5.05 (d, J = 3.6 Hz, 1 H, H-1a), 4.98 (d, J =

3.6 Hz, 1 H, H-1b), 4.88 (d, J = 12.0 Hz, 1 H, CHH), 4.80 (d, J = 10.8 Hz, 1 H, CHH), 4.72 (d, J = 11.2 Hz, 1 H, CHH), 4.63 (d, J = 11.2 Hz, 1 H, CHH), 4.54 (bd, 3 H,3 CHH), 4.47 (d, J = 10.8 Hz, 1 H, CHH), 4.36 (dd, J = 5.2, 9.2 Hz, 1 H, H-5a), 4.28 (d, J = 2.8 Hz, 1 H, H-4a), 4.10 (bs, 1 H, H-4b), 4.03-3.85 (m, 8 H, H-6ba, 5b, 3b, 3a, 2b, 2a,

H-1º), 3.60 (dd, J = 3.6, 11.2 Hz, 1H, H-2a), 3.56-3.49 (m, 2 H, H-6bb, H-6aa), 3.14-3.09 (m, 2 H, H-6ab), 1.20 (d, J = 6.0

Hz, 1 H, CH3), 1.19 (d, J = 6.0 Hz, 1 H, CH3). 13C-APT (CDCl3, 100 MHz) δ 138.65, 137.99, 137.70, 137.61 (aromatic

C), 128.64, 128.58, 128.55, 128.38, 128.32, 128.16, 128.12, 128.09, 128.01, 127.90, 127.87, 127.75, 127.74, 127.64,

127.25 (aromatic CH), 98.24 (C-1b), 96.84 (C-1a), 77.35 (C-3b), 75.94 (C-3a), 75.03, 73.67, 73.22 (PhCH2), 73.04

(C-4b), 72.86 (C-4a), 71.91, 71.87 (PhCH2), 71.01 1º), 69.37 5b), 69.20 5a), 67.74 6a), 67.18 6b), 60.35

(C-2b), 59.51 (C-2a), 23.36 (CH3), 21.68 (CH3). HR-MS: Calculated for C50H56O9N6 [M+NH4]+: 902.44470, found:

902.44467.

Synthesis of disaccharide 23: Donor 15 (5 g, 8.2 mmol) and acceptor 19 (3.32 g, 6.95 mmol) (donors and acceptors co-evaporated with toluene three times) were dissolved in DCM (65 mL), cooled to 0 oC and TfOH (60 μL) was added. The reaction was stirred at 0 ℃ until

TLC-analysis showed complete conversion of the donor. The reaction was quenched with Et3N after completed checking by TLC, filtered and concentrated in vacuo. Compound 23

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colorless syrup. [α]D20 +153.3 (c=1, CHCl3). IR (neat, cm-1) ν 651, 698, 738, 797, 826, 984, 1043, 1066, 1100, 1171, 1364,

1473, 2107 (N3), 2859, 2933. 1H-NMR (CDCl3, 400 MHz) δ 7.53-7.51 (m, 2 H, aromatic H), 7.44-7.22 (m, 18 H, aromatic

H), 5.64 (d, J = 3.6 Hz, 1 H, H-1b), 5.59 (d, J = 5.2 Hz, 1 H, H-1a), 5.00 (d, J = 10.4 Hz, 1 H, CHH), 4.91 (d, J = 10.4

Hz, 1 H, CHH), 4.73 (d, J = 11.6 Hz, 1 H, CHH), 4.63 (d, J = 11.6 Hz, 1 H, CHH), 4.43-4.37 (m, 4 H, 2 CHH, 4b, H-5a), 3.96-3.77 (m, 6 H, H-2a, H-2b, H-3a, H-4a, H-6), 3.72-3.64 (m, 2 H, H-3b, H-6a), 3.53 (dd, J1 = 2.0 Hz, J2 = 10.8

Hz, 1 H, H-6b), 3.42 (s, 1 H, H-5b), 1.03 (s, 9 H, 3 CH3), 0.97 (s, 9 H, 3 CH3). 13C-APT (CDCl3, 100 MHz) δ 137.91,

137.78, 137.42, 133.48 (aromatic C), 132.14, 129.17, 128.62, 128.58, 128.51, 128.48, 128.00. 127.98, 127.79, 127.54 (aromatic CH), 97.66 (C-1b), 87.10 (C-1a), 82.33 (C-3a), 75.54 (C-3b), 74.99, 73.33 (PhCH2), 72.81 (c-4a), 71.27 (c-5a),

70.45 (PhCH2), 69.55 (C-4b), 68.89 (C-6), 67.97 (C-5b), 66.89 (C-6), 64.61 (C-2a), 58.14 (C-2b), 27.66 (3 CH3), 27.28

(3 CH3), 23.40, 20.73. HR-MS: Calculated for C47H58N6O8SSi [M+NH4]+: 912.41444, found: 912.41409.

N-phenyl trifluoroacetimidate disaccharide donor 26: Compound 30 (4.15 g, 4.21 mmol)

was dissolved in acetone:H2O (10:1, 44 mL). N-Iodosuccinimide (NIS) (2.0 g, 8.8 mmol)

was added in one portion and the reaction was stirred at room temperature for 2 hours. The solution was diluted with DCM and the reaction was quenched with saturated aqueous Na2S2O3. Then the organic layer was washed with water and brine. The organic layer was dried with anhydrous

MgSO4, filtered and concentrated in vacuo, and the product purified by column chromatography (pentane : ethyl acetate

(EA) = 3:1). The lactol 31 was obtained as colourless syrup. Next, the lactol was dissolved in acetone (40 mL). Cs2CO3

(1.9 g) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (960 μL) were added to the solution respectively. The reaction was stirred overnight, then quenched with Et3N, filtered and concentrated in vacuo. The product was purified by column

chromatography (pentane:EA = 40:1-20:1). Compound 26 (3.7 g, 83% over two steps) was obtained as yellow syrup. IR (neat, cm-1) ν 695, 734, 818, 1027, 1116, 1209, 1312, 1454, 1489, 1497, 1717, 2107, 2870, 2918. 1H-NMR (CDCl 3, 500 MHz) δ 7.80-7.09 (m, aromatic H), 6.81 (bt, 1 H), 5.65 (dd, 1 H), 5.01-4.87 (m), 4.68-4.54 (m), 4.45-4.42 (m), 4.33-4.18 (m), 4.03-3.41 (m). 13 C-APT (CDCl 3, 125 MHz) δ 143.37, 143.22, 138.20, 138.18, 137.78, 137.58, 137.55, 137.53, 135.58, 133.26, 133.16 (aromatic C), 128.91, 128.67, 128.53, 128.52, 128.44, 128.43, 128.28, 128.08, 128.05, 128.00, 127.97, 127.92, 127.84, 127.81, 127.69, 127.66, 127.65, 127.55, 127.20, 127.17, 124.67, 124.56 (aromatic CH), 119.41 (C-1), 98.34 (C-1), 98.19 (C-1), 83.55, 80.97, 77.55, 77.25, 75.51, 75.18, 75.16, 74.94, 74.89, 73.64, 73.60, 73.29, 73.21, 73.11, 73.02, 72.76, 72.69, 72.30, 72.16, 70.42, 70.34, 68.97, 68.52, 65.80, 63.65, 59.74, 59.64. HR-MS: Calculated for C59H56F3N7O9 [M-[O(C=NPh)CF3]+OH+Na]+: 910.41340, found: 910.41374.

Synthesis of disaccharide acceptor 27: The compound 33 (865 mg, 1.0 mmol) was dissloved in CH3CN (10 Ml). Then BnBr (182 μL), borinic acid-catalyzed (22 mg),

K2CO3 (148 mg), KI (166 mg), were added in the mixture. The reaction stirred at 60 oC

until TLC-analysis indicated full consumption of the starting material (24 h). Then the mixture was diluted with ethyl acetate and the reaction quenched with saturated NaHCO3. The organic phase was washed with water and brine, dried with anhydrous MgSO4, filtered and concentrated

in vacuo. The product was purified by silica gel column chromatography. Compound 27 (910 mg, 94%) was obtained as

a colorless syrup. [α]D20 +66.6 (c=1, CHCl3). IR (neat, cm-1) ν 697, 736, 1040, 1096, 1259, 1455, 1734 (C=O), 2106 (N3),

2869, 2928. 1H-NMR (CDCl

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H, PhCH2), 4.96 (d, J = 10.4 Hz, 1 H, CHH), 4.91 (d, J = 3.6 Hz, 1 H, H-1a), 4.88 (d, J = 10.4 Hz, 1 H, CHH), 4.63 (bs,

2 H, 2 CHH), 4.55 (d, J = 12.0 Hz, 1 H, CHH), 4.45 (d, J = 12.0 Hz, 1 H, CHH), 4.47-4.36 (m, 3 H, 3 CHH), 4.07-4.03 (m, 2 H, H-3a, H-5a), 3.91-3.44 (m, 12 H), 3.32 (dd, J1 = 11.2 Hz, J2 = 3.6 Hz, 1 H, H-2a), 2.65 (s, 1 H, OH), 2.38 (t, J =

7.6 Hz, 2H, H-5o), 1.73-1.63 (m, 4 H, H-2º, H-4º), 1.47-1.39 (m, 2 H, H-3º). 13C-APT (CDCl

3, 100 MHz) δ 173.54 (C=O),

138.32, 137.81, 137.27, 136.14 (aromatic C), 129.07, 128.72, 128.65, 128.54, 128.50, 128.39, 128.28, 128.01, 127.89, 127.82, 127.63, 127.56, 127.32 (aromatic CH), 97.92 (C-1b), 97.68 (C-1a), 80.79 (C-3a), 76.25 (C-3b), 74.46 (PhCH2),

73.94 (C-4a), 73.76, 73.28, 71.68 (PhCH2), 70.10 (C-4b), 69.52, 69.44 (C-6), 69.16 (C-5b), 68.20 (C-1º), 66.44 (C-5a),

66.23 (PhCH2), 63.74 (C-2a), 58.88 (C-2b), 34.24 (C-5º), 29.13 (C-2º), 25.74 (C-3º), 24.76 (C-4º). HR-MS: Calculated

for C53H60N6O11 [M+NH4]+: 974.46583, found: 974.46660.

Synthesis of disaccharide 29: Compound 23 (4.1 g, 4.6 mmol) was dissoveld in THF (40 mL) in a round flusk. Then HF-pyridine (1.2 mL) was added in the solution. The reaction stirred until TLC-analysis indicated full consumption of the starting material (30 min). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3. The

organic phase was washed with water and brine, dried with anhydrous MgSO4, filtered and concentrated in vacuo. The

crude compound 28 was dissloved in CH3CN (47 mL). Then BnBr (880 μL), borinic acid-catalyzed (110 mg), K2CO3

(710 mg), KI (800 mg) were added in the mixture. The reaction stirred at 60 oC until TLC-analysis indicated full

consumption of the starting material (24 h). Then the mixture was diluted with ethyl acetate and the reaction quenched with saturated NaHCO3. The organic phase was washed with water and brine, dried with anhydrous MgSO4, filtered and

concentrated in vacuo. The product was purified by silica gel column chromatography. Compound 29 (3.6 g, 94% yield with two steps) was obtained as a colorless syrup. [α]D20 +11.7 (c=1, CHCl3). IR (neat, cm-1) ν 697, 737, 1029, 1046, 1077,

1266, 2106 (N3), 2870, 2919, 3493. 1H-NMR (CDCl3, 400 MHz) δ 7.54-7.51 (m, 2 H, aromatic H), 7.41-7.23 (m, 23 H, aromatic H), 5.61 (d, J = 3.6 Hz, 1 H, H-1b), 5.60 (d, J = 5.2 Hz, 1 H, H-1a), 5.00 (d, J = 10.4 Hz, 1 H, CHH), 4.94 (d, J = 10.4 Hz, 1 H, CHH), 4.66 (s, 2 H, PhCH2), 4.50-4.37 (m, 3 H, 2 CHH, 5a), 4.08 (s, 1 H, 4b), 3.97-3.91 (m, 2 H, H-2b, H-4a), 3.85-3.37 (m, 5 H), 3.66 (dd, J1 = 2.4 Hz, J2 = 10.8 Hz, 1 H, H-6b), 3.59 (dd, J1 = 5.6 Hz, J2 = 9.6 Hz, 1 H), 3.51 (dd, J1 = 5.6 Hz, J2 = 9.6 Hz, 1 H), 2.63 (s, 1 H, OH). 13C-APT (CDCl3, 100 MHz) δ 138.26, 137.83, 137.64, 137.24, 133.50 (aromatic C), 132.29, 128.77, 128.63, 128.53, 128.39, 128.34, 128.08, 127.97, 127.91, 127.86, 127.85, 127.63, 127.55 (aromatic CH), 98.17 (C-1b), 87.10 (C-1a), 82.14 (C-3a), 76.29 (C-3b), 75.14 (PhCH2), 74.36 (c-4a), 73.78, 73.19,

71.77 (PhCH2), 71.29 (c-5a), 69.52 (C-6), 69.40 (C-6), 69.30 (C-5b), 66.47 (C-4b), 64.75 (C-2b), 58.96 (C-2a). HR-MS:

Calculated for C46H48N6O8S [M+NH4]+: 862.35926, found: 862.35895.

Synthesis of thio-disaccharide 30: The compound 29 (3.83 g, 4.53 mmol) was dissloved in DMF (10 mL). Then NaH (544 mg) and NapBr (1.5 g) were added in the mixture. The reaction stirred until TLC-analysis indicated full consumption of the starting material (2 h). Then the mixture was diluted with ethyl acetate and the reaction quenched with ice water. The organic phase was washed with water and brine, dried with anhydrous MgSO4, filtered and

concentrated in vacuo. The product was purified by silica gel column chromatography. Compound 30 (4.15 g, 93% yield) was obtained as a colorless syrup. [α]D20 +208.6 (c=1, CHCl3). IR (neat, cm-1) ν 697, 737, 1028, 1049, 1093, 1123, 1362,

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H-1b), 5.59 (d, J = 5.2 Hz, 1 H, H-1a), 5.01-4.89 (m, 3 H, 3 CHH), 4.70-4.62 (m, 3 H, 3 CHH), 4.50 (d, J = 12.0 Hz, 1 H, CHH), 4.44-4.40 (m, 2 H, CHH, 5b), 4.27 (d, J = 11.6 Hz, 1 H, CHH), 4.17 (d, J = 11.6 Hz, 1 H, CHH), 4.04 (s, 1 H, H-4b), 3.97-3.81 (m, 6 H, H-2a, H-2b, H-3a, H-3b, H-4a, H-5a), 3.77-3.65 (m, 2 H, H-6), 3.51-3.40 (m, 2 H, H-6). 13

C-APT (CDCl3, 100 MHz) δ 138.31, 137.79, 137.60, 137.58, 135.58, 133.56, 133.24, 133.15 (aromatic C), 133.28, 129.18,

128.67, 128.64, 128.50, 128.37, 128.27, 128.07, 127.99, 127.97, 127.93, 127.80, 127.56, 127.44, 127.20, 126.49, 126.21, 126.09 (aromatic CH), 98.34 (C-1b), 87.13 (C-1a), 82.24 (C-3a), 77.52 (C-3b), 75.20, 74.91 (PhCH2), 74.11 (c-4a), 73.58,

73.11 (PhCH2), 72.77 (C-4b), 72.27 (PhCH2), 71.28 (C-5b), 70.36 (C-5a), 69.42 (C-6), 68.56 (C-6), 64.80 (C-2a), 69.75

(C-2b). HR-MS: Calculated for C57H56N6O8S [M+NH4]+: 1002.42186, found: 1002.42125.

Synthesis of disaccharide 32: Donor 15 (1.09 g) and acceptor 7 (790 mg) (donors and acceptors co-evaporated with toluene three times) were dissolved in DCM (12 mL), cooled to 0 oC and TfOH (12 μL) was added. The reaction was stirred at 0 ℃ until

TLC-analysis showed complete conversion of the donor. The reaction was quenched with Et3N after completed checking by TLC, filtered and concentrated in vacuo.

Compound 32 (1.24 g, 92% yield) was obtained with full α-selectivity as a colorless syrup. [α]D20 +95.9 (c=1, CHCl3). IR (neat, cm-1) ν 651, 698, 737, 765, 797, 826, 984, 1004, 1040, 1130, 1144, 1171, 1455, 1474, 1735 (C=O), 2106 (N3), 2860, 2933. 1H-NMR (CDCl3, 400 MHz) δ 7.44-7.24 (m, 20 H, aromatic H), 5.67 (d, J = 3.6 Hz, 1 H, H-1b), 5.12 (s, 1 H, PhCH2), 4.96 (d, J = 10.4 Hz, 1 H, CHH), 4.92 (d, J = 3.6 Hz, 1 H, H-1a), 4.86 (d, J = 10.4 Hz, 1 H, CHH), 4.71 (d, J = 11.6 Hz, 1 H, CHH), 4.61 (d, J = 11.6 Hz, 1 H, CHH), 4.48 (s, 2 H, PhCH2), 4.36 (d, J = 2.0 Hz, 1 H, H-4b), 4.06 (dd, J1 = 10 Hz, J2 = 8.4 Hz, 1 H, H-3a), 3.91-3.78 (m, 4 H), 3.74-3.45 (m, 6 H), 3.34-3.30 (m, 2 H, H-2a, H-1ºb), 2.39 (t, J = 7.6 Hz, 2H, H-5o), 1.74-1.64 (m, 4 H, H-2º, H-4º), 1.48-1.42 (m, 2 H, H-3º), 1.03 (s, 9 H,

3 CH3), 0.95 (s, 9 H, 3 CH3). 13C-APT (CDCl3, 100 MHz) δ 173.54 (C=O), 138.03, 137.85, 137.67, 136.15 (aromatic C),

128.66, 128.56, 128.30, 128.02, 127.96, 127.92, 127.86, 127.60 (aromatic CH), 97.77 (C-1b), 97.47 (C-1a), 80.97 (C-3a), 75.51 (C-3b), 74.25, 73.52 (PhCH2), 72.43 (c-4a), 70.45 (PhCH2), 70.11 (c-5a), 69.57 (C-4b), 69.10 (C-6), 68.25 (C-1º),

67.90 (C-5b), 66.92 (C-6), 66.25 (PhCH2), 63.61 (C-2a), 58.13 (C-2b), 34.25 (C-5º), 29.15 (C-2º), 27.70 (3 CH3), 27.31

(3 CH3), 25.76 (C-3º), 24.76 (C-4º), 23.44, 20.75. HR-MS: Calculated for C54H70N6O11Si [M+NH4]+: 1024.52101, found:

1024.52157.

Synthesis of disaccharide 33: Compound 32 (1.16 g, 1.15 mmol) was dissoveld in THF (11 mL) in a round flusk. Then HF-pyridine (300 μL) was added in the solution. The reaction stirred until TLC-analysis indicated full consumption of the starting material (30 min). Then the mixture was diluted with DCM and the reaction quenched with saturated NaHCO3. The organic phase was washed with water and brine, dried with

anhydrous MgSO4, filtered and concentrated in vacuo. The crude compound 28 was dissloved in CH3CN. The product

was purified by silica gel column chromatography. Compound 33 (910 mg, 91% yield) was obtained as a colorless syrup. [α]D20 +80.6 (c=1, CHCl3). IR (neat, cm-1) ν 698, 738, 1040, 1145, 1262, 1354, 1455, 1733 (C=O), 2106 (N3), 2872, 2932,

3461. 1H-NMR (CDCl

3, 400 MHz) δ 7.38-7.25 (m, 20 H, aromatic H), 5.65 (d, J = 3.6 Hz, 1 H, H-1b), 5.12 (s, 1 H,

PhCH2), 4.97 (d, J = 10.4 Hz, 1 H, CHH), 4.92 (d, J = 3.6 Hz, 1 H, H-1a), 4.88 (d, J = 10.4 Hz, 1 H, CHH), 4.67-4.54 (m,

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